Whey proteins (20% of cow's milk proteins)
Alpha-lactalbumin (Bos d 4)	Role in milk allergy is controversial and prevalence data across studies vary between 0-80% of patients reacting to this protein.
Beta-lactoglobulin (Bos d 5)	Most abundant cow’s milk whey protein; it occurs in the milk of many other species but is not present in human milk. 13-76% of patients are found to react to this protein.
Bovine serum albumin (Bos d 6)	Involved in other allergies such as beef; it accounts for between 0 and 88% of sensitization events, while clinical symptoms occur in up to 20% of patients. Sensitization to Bos d 6 is independent of sensitization to the other milk allergens. Due to cross-reactivity between bovine serum albumin and beef, almost all children allergic to beef are allergic to milk; 10-20% of children allergic to milk are allergic to beef.
Immunogobulin (Bos d 7) --	Seldom responsible for clinical symptoms in milk allergy.
Caseins (80% of cow's milk protein)
Caseins (Bos d 8)	Casein allergens are collectively labeled Bos d 8 Consist of alpha-s1, alpha-s2, beta, gamma, and kappa casein which share little sequential homology. Despite this, simultaneous sensitization to these caseins is frequently observed. Patients are more often sensitized to alpha (100%) and kappa caseins (91.7%).

Note

Most patients are sensitized to several different cow's milk proteins
- Co-sensitization to the major milk allergens (casein, beta-lactoglobulin, and alpha-lactalbumin) is common.
- Co-sensitization to minor milk allergens (including bovine lactoferrin, BSA, and immunoglobulins) is also common; seen in up to half of patients and some are sensitized exclusively to these minor allergens.

Effects of heating on allergenicity
- Reduction in allergenicity by destroying conformational epitopes
- Heating of b-lactoglobulin results in the formation of intermolecular disulfide bonds and subsequent binding to other food proteins, making b-lactoglobulin less allergenic
- Heating decreases allergenicity of whey proteins (particularly beta-lactoglobulin) presumably by denaturation that results in loss of conformational epitopes.
  - Patients exclusively allergic to whey proteins usually tolerate yogurt and baked goods containing milk
- Patients with transient milk allergy produce IgE antibodies primarily directed at conformational epitopes, whereas those with persistent allergy produce IgE antibodies against sequential epitopes, which are heat stable
- In the case of beef which contains bovine serum albumin, industrial treatment (freeze-dried, homogenized) more than heating may modify the allergic reactivity of this meat in beef-allergic children. Cooked processed beef may be safer alternative to raw meat cooked at home
- Aged parmigiano reggiano cheese appears to have intact levels of beta-lactoglobulin but significantly lower levels of casein due to enzymatic degradation
Mammalian milk contains alpha-gal

Cow's Milk Allergen Cross-reactivity

Diagnosis

Skin Prick Test

SPT with positive defined as wheal ≥3 mm	Sensitivity-----	Specificity-----
Combined	67	74
Eczema excluded	71	73
Children suspected of milk allergy <12 months old-----	55	75
Children suspected of milk allergy >12 months old	81	75

Specific IgE

sIgE with positive defined as ≥0.35 IU/L	Sensitivity-----	Specificity-----
Combined	72	57
Eczema excluded	62	62
Children suspected of milk allergy <12 months old-----	77	52
Children suspected of milk allergy >12 months old	52	71

Note

Phadia ImmunoCAP sIgE component testing available for:
- Whey
  - nBos d 4 α-lactalbumin
  - nBos d 5 ß-lactoglobulin
  - Lactoferrin
- Casein - nBos d 8

Phadia ImmunoCAP ISAC microarray panel includes:
- nBos d 4
- nBos d 5
- nBos d 6 BSA
- nBos d 8
- Lactoferrin

Testing Algorithm (DRACMA)

Patient type	SPT	sIgE
High pretest probability (80%) Includes patients who experienced an anaphylactic reaction in the past.	Use SPT with a positive value ≥3 mm as a triage test to avoid oral food challenge in patients with a positive test This approach results in 5-6% false positive rate	Use sIgE with a cut-off of ≥0.7 IU/L as a triage test to avoid oral food challenge in patients with a positive test This approach results in 5% false positive rate
Average pretest probability (40%) Estimated based on the history and would represent the majority of situations.	Use OFC as the only test (without performing SPT or sIgE)
Low pretest probability (10%) Estimated based on the history and would include (for example) patients with unexplained gastrointestinal symptoms like GERD	Use SPT with a positive value ≥3 mm as a triage test to avoid oral food challenge in patients with a negative test This approach results in 2-4% false negative rate	Use sIgE with cut-off ≥0.7 as a triage test to avoid oral food challenge in patients with a negative test This approach results in 2-5% false negative rate

Evaluation of cross-reactive foods

Cow's milk BSA - Beef albumin cross-reactivity
- Almost all children allergic to beef are allergic to milk; 10-20% of children allergic to milk are allergic to beef.
- Industrial treatment (freeze-dried, homogenized) more than cooking beef well may modify the allergic reactivity of this meat in beef-allergic children. Cooked processed beef may be safer alternative to raw meat cooked at home.
- Total avoidance of beef by all cow’s milk-allergic children is not justified. In this setting "an allergist’s evaluation of cross-sensitization makes sense during the diagnostic work-up of milk allergy."

Evaluation for alpha-gal allergy

New onset milk allergy in patient >5 years old with history of tick bite in the lone star tick endemic region (southern New England coast, Mid-Atlantic, Southeast and Gulf states) shoud raise suspicion for alpha-gal allergy and a sIgE to alpha-gal may be checked

Monitoring Tests Over Time

Cow's Milk Allergy Phenotypes

75% of children with cow's milk allergy (with sIgE <35 kU/L) tolerate baked foods containing milk
- Examples of baked foods containing milk used in clinical studies include:
  - Muffin containing 1.3 g of milk protein (nonfat dry milk powder; Nestle Carnation) baked at 350F for 30 min
  - Waffle (<0.625 inches thick to ensure thorough heating), containing 1.3 g of milk protein (nonfat dry milk powder; Nestle Carnation) cooked in a waffle maker at ~500F for 3 min
  - Pizza (Amy’s Cheese Pizza, Amy's Kitchen, Inc), containing 4.6 g of milk protein, baked at 425F for 13 min or longer.

Patient type	Clinical outcome
Heated-milk tolerant, unheated milk intolerant	More likely to have transient milk allergy Over a median of ~3 years, much more likely (28 times) to tolerate unheated milk vs. children that do not tolerate heated milk Continued ingestion of foods containing heated milk accelerates resolution of milk allergy (via immunologic changes similar to oral immunotherapy)
Heated milk intolerant	More likely to have a persistent milk allergy
Heated and unheated milk tolerant	Clinical milk allergy resolved or never present

Casein specific IgE <0.7 kUA/L is a very favorable prognostic factor for tolerance of baked milk with the vast majority of kids tolerating baked milk with such level (Wegrzyn)
Low casein specific IgE level is a very favorable prognostic factor for tolerance of baked milk, with casein ImmunoCAP sIgE >20 unlikely to pass a baked milk challenge, and level <0.94 indicate a very low risk of reacting to baked milk

In one study, 58% of children with challenge proven cow's milk allergy tolerated aged parmigiano reggiano cheese (13.3 grams, aged 36 months), with lower levels of sensitivity to beta-lactoglobulin noted as a favorable factor
- Aged parmigiano reggiano cheese appears to have intact levels of beta-lactoglobulin but significantly lower levels of casein due to enzymatic degradation
- Challenge procedure:
  
  13 g of PR = 200 mL of cow' milk in protein content

Oral Food Challenge Procedure to Cow's Milk (Baked and Unbaked)

Treatment

Strict avoidance of cow's milk
- Parents to read food ingredient labels
- Some drugs/vaccines reported to be contaminated with milk protein and can result in adverse reactions in severely milk-allergic patients
  - Dry powder inhalers, certain forms of singulair, claritin, benadryl, etc
  - Lactulose, a laxative derived from lactose
  - Some concentrations of IV methylprednisolone (Pfizer Solu-medrol) and tablets
  - Vaccines containing diphtheria, tetanus, and acellular pertussis may contain nanograms of casein and are reported to cause reactions in children with severe milk allergy. Caution is advised when administering these vaccines to these children (Kelso: give full dose but observe for 30 minutes)
- Replace cow's milk in diet:

Children <1 year
History of anaphylaxis	Breastfeeding with strict elimination of cow's milk protein from mother's diet Use amino acid formula, e.g. Nutramigen AA, Neocate, Elecare infant formula
Low risk of anaphylaxis No prior history of anaphylaxis, or Currently tolerating EHF	Breastfeeding with strict elimination of cow's milk protein from mother's diet Use extensively hydrolyzed casein infant formula (EHF), e.g. Nutramigen, Alimentum. Pregestemil is an EHF that is typically used for patients with fat malabsorption, such as CF, short bowel syndromes, etc
Note that soy or rice infant formulas (vs. AA or extensively hydrolyzed casein formula) not recommended by DRACMA guidelines due to lack of sufficient evidence.

Children >1 year
History of anaphylaxis	Breastfeeding with strict elimination of cow's milk protein from mother's diet Amino acid toddler formula, e.g. Neocate Junior, Elecare Jr unflavored and vanilla Consider alternative enriched milk beverages (soy, rice, oat, almond, potato) Soy milk similar in nutrition to 2% cow's milk but be aware of co-existing soy allergy (~15% of children with cow's milk allergy)
Low risk of anaphylaxis No prior history of anaphylaxis, or Currently tolerating EHF	Consider: Breastfeeding with strict elimination of cow's milk protein from mother's diet Amino acid toddler formula (e.g. Neocate Junior, Elecare Jr unflavored and vanilla) Extensively hydrolyzed infant formula - contains approximately half the protein and calcium/Vit D of whole milk Alternative enriched milk beverages (soy, rice, oat, almond, potato) Soy milk similar to nutrition to 2% cow's milk but be aware of co-existing soy allergy (~15% of children with cow's milk allergy) Alternative mammalian milks that have minimal cross-reactivity with cow's milk proteins are not readily available in US (camel, horse, donkey)

Supplement diet with foods containing protein, fat, calcium, and vitamins primarily obtained from cow's milk in a child's diet (excluding foods that are not age-appropriate or that the child is allergic to):
- Protein from meat, fish, poultry, eggs, soy products, peanut, other legumes, tree nuts and seeds
- Fat from vegetable oils, margarine, avocado, meats, fish, poultry, peanut, tree nuts, seeds
- Calcium from enriched alternative milk beverages (soy, rice, oat, almond, hemp, potato), calcium fortified tofu, calcium fortified juice
- Vitamin D from enriched alternative milk beverages, fortified margarine, eggs, fish oils
- Vitamin B12 from meat, fish, poultry, eggs, enriched alternative milk beverages
- Vitamin A from liver, egg yolk, fortified margarine, dark green leafy vegetables, deep orange fruits and vegetables, enriched alternative milk beverages
- Pantothenic acid (vitamin B5) from meat, vegetables, eggs, whole grains, legumes, fish
- Riboflavin from dark green leafy vegetables, enriched and whole grain products

If patient tolerated cow's milk protein in heated/processed forms (e.g. baked items) but not in less processed forms (drinking milk), consider allowing continued ingestion of milk in its heated/processed forms, with a warning regarding under-cooking and ingestion of large quantities.
- E.g. if tolerated baked food with milk, consume 1-3 servings per day of store-bought baked milk products with milk listed as 3rd or lower ingredient or home-baked products with an equivalent amount of milk protein. If tolerated baked cheese pizza, eat any brand of well-cooked cheese pizza 4-7 times weekly and limited to 1 daily serving.